Human immunodeficiency virus (HIV) binds and enters cells via interaction of its envelope glycoprotein gp120 with CD4 and either the CCR5 or the CXCR4 chemokine co-receptor. Binding of CD4 to gp120 causes a structural change in the envelope complex which exposes the chemokine binding domains of gp120. This binding induces fusion of the viral and target cell membranes.
HIV-1 affinity for CD4 and CCR5 is associated with differential pathogenicity. Therefore, a dually inducible cell line based system that quantitatively and comprehensively characterizes viral entry efficiency as a co-dependent function of CD4 and CCR5 expression levels has been produced. This receptor affinity profiling system (Affinofile) has revealed biologically relevant phenotypes in viral envelope proteins (envs) with differential CD4/CCR5 usage efficiencies. For example, Elite Suppressor envs have shown reduced efficiency of CD4/CCR5 usage compared to Chronic Progressor envs, and for vicriviroc-resistant envelopes, the degree of resistance is a function of the level of CCR5 cell surface density.
One method of determining HIV receptor usage is through HIV indicator cell lines. Several HIV indicator cell lines are currently available. However, all HIV indicator cell lines currently available use the increased production of a fluorescent protein or a (firefly) luciferase protein as an indication of infection. The use of only a fluorescent protein would require the laborious preparation of the cells and examining of cells on a fluorescent detection system. Alternatively, the use of nonsecreted luciferase would require careful attention to non-trivial matters such as complete lysis of cells before assessment of luciferase activity. Moreover, GFP or firefly (or even Renilla-luciferase) luciferase detection are endpoint assays, since the cells have to be fixed or lysed to assess HIV infection.
Additionally, all indicator cell lines express the receptor (CD4) and co-receptor (CCR5) for HIV at fixed, often over-expressed supraphysiological levels. This hinders the utility of those cell lines because different HIV isolates have varying capacity to use the receptor and co-receptor found at different expression levels on various cell types in vivo. Accordingly, the need exists in the field for an improved method for detecting pseudotype and/or replication competent HIV, from cloned or uncloned isloates, in cell media or human serum.